Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. This is a particularly challenging concept to investigate, since there is no straightforward way a priori to identify which cells may be susceptible or resistant to transformation prior to clonal expansion. We propose an innovative series of experiments to test the hypothesis that pre-existing epigenetic heterogeneity among normal cells affects the likelihood of malignant transformation and further progression upon genetic alteration of a cancer driver gene. In Specific Aim 1, we will use an improved protocol for single-cell whole genome bisulfite sequencing to document the degree of DNA methylation heterogeneity among flow-sorted mouse colon stem cells. For the first time, this will provide insight into whether low-level DNA methylation abnormalities observed in normal tissues are stochastically distributed, or whether subsets of stem cells bear multiple concerted epigenetic abnormalities. In Specific Aim 2, we will first generate large numbers of individual clonally expanded mouse colon organoids harboring conditional alleles for Apc, Trp53, Kras and Braf. Each organoid will be cryopreserved and concurrently subjected to whole genome bisulfite sequencing to delineate each methylome. Organoids representing diverse methylomes will be thawed, the cancer drivers activated, and then tested for tumorigenicity by colon enema engraftment. This innovative experiment aspires to provide the first direct evidence for the contribution of pre-existing epigenetic heterogeneity to cancer predisposition. In Specific Aim 3, we will provide empirical evidence for this concept by using a novel in vivo transcriptional control system to transiently up- or down-regulate DNA methyltransferase activity in mice, prior to activation of conditional key cancer drivers. This aim should provide a proof of concept for a causal role of pre-existing epigenetic variation affecting cancer propensity. This proposal addresses a concept for which there is considerable indirect evidence, but which remains a poorly studied area because of the technical and conceptual challenges presented by the premise. Our proposed experiments are intricate and complex, but we have considerable expertise in all areas of the proposal, and have developed cutting-edge solutions to many of the technical hurdles. We may not be able to conclusively delineate in detail all epigenetic variations that predispose to malignancy, but this exploratory project should provide a proof of principle for the importance of pre-existing epigenetic heterogeneity in cancer susceptibility. The outcome of this study should have a substantial impact on our understanding of lifetime accumulation of cancer risk, with implications for cancer screening and prevention.